Glycated hemoglobin is a glycation product of hemoglobin in which glucose is bound thereto. Hemoglobin takes a tetrameric structure consisting of α and β chains. The glycated product of the N terminus of β chain of hemoglobon is called hemoglobin A1c, which increases with increase in blood glucose level and as such, is routinely measured as a diabetes mellitus marker for monitoring glycemic control in clinical laboratory examinations.
Known methods for measuring glycated hemoglobin include, for example, chromatography such as HPLC, electrophoresis, antibody-based immunoassay such as latex immunoagglutination assay, and enzymatic assay using an enzyme reactive to a glycated protein and an enzyme reactive to a glycated peptide and/or a glycated amino acid.
A known method for enzymatically measuring glycated hemoglobin comprises: first denaturing glycated hemoglobin in a hemoglobin-containing sample using a denaturant; reacting the denatured glycated hemoglobin with a proteolytic enzyme; subsequently reacting the generated glycated peptide with glycated peptide oxidase; reacting the generated hydrogen peroxide with a chromogen capable of developing color by oxidation in the presence of a peroxidatively active substance such as peroxidase to convert the chromogen to a dye; and measuring the glycated hemoglobin on the basis of the absorbance of the generated dye. In this context, the denaturant is used for reacting glycated hemoglobin with the proteolytic enzyme. Previously known denaturants are: a compound having an acetic acid group or a salt thereof, N-acyl taurine or a salt thereof, and polyoxyethylene alkyl ether sulfuric acid or a salt thereof (Patent Document 1); anionic surfactants such as polyoxyethylene alkyl ether sulfates and polyoxyethylene alkylphenyl ether sulfates (Patent Document 2); sodium lauryl sulfate and sulfonic acid compounds such as sodium dodecyl benzene sulfonate (Patent Document 3); sulfonic acid compounds and nitro compounds (Patent Document 4); quaternary ammonium salts, alkyl benzyl dimethyl ammonium chloride, lauryl dimethylamine oxide, and the like (Patent Document 5).
In a method for enzymatically measuring glycated hemoglobin, a method is often used which involves: converting glycated hemoglobin to hydrogen peroxide; reacting the generated hydrogen peroxide with a chromogen capable of developing color by oxidation in the presence of a peroxidatively active substance such as peroxidase to convert the chromogen to a dye; and measuring the glycated hemoglobin on the basis of the absorbance of the generated dye. In this context, a leuco chromogen is often used as the chromogen capable of developing color by oxidation. The leuco chromogen is a chromogen that generates a dye through reaction with hydrogen peroxide in the presence of a peroxidatively active substance such as peroxidase. This type of chromogen, unlike coupling-type chromogens, generates a dye in itself and known leuco chromogens include, for example, phenothiazine leuco chromogens, triphenylmethane leuco chromogens, and diphenylamine leuco chromogens (see e.g., Patent Documents 6 to 8).
The leuco chromogen is preferably used as a highly sensitive chromogen in the quantification of an analyte component, such as glycated hemoglobin, which is contained only in a trace amount in a sample (see e.g., Non-patent Document 1). The leuco chromogen, however, has poor preservation stability and undesirably develops color spontaneously with time, particularly, in a solution. The poor stability of this leuco chromogen disadvantageously causes the inaccurate measurement of an analyte component in a sample.
To cope with this undesirable poor stability of the leuco chromogen, methods for stabilizing the leuco chromogen in a solution have been studied and reported so far (see e.g., Patent Documents 9 and 10).